A high performance liquid chromatography investigation of the urobilinoids

Abstract

This investigation was concerned with the detection of the urobilinoids (i.e. urobilinogen) by High Performance Liquid Chromatography (HPLC) using a Reversed Phase column. Attempts were unsuccessfully made to crystallize the urobilinoids in order to provide a convenient sample supply for HPLC studies.

Cyclic voltammograms for urobilinogen and the mobile phase (.1 M KCl in 20% MeOH) were taken in order to determine the working potential for the HPLC electrochemical detector. Electrochemical detection was not possible due to the high polarity of the aqueous solvents required by the detector. Absorption spectra indicated that UV detection was possible Urobilinogen and bilirubin samples were injected onto the column using various MeOH and MeOH/acetonitrile solvent systems. A separation of bilirubin and urobilinogen was obtained by using a 50% MeOH mobile phase. Bilirubin was not retained on the column for the MeOH/acetonitrile systems, although excellent retention of urobilinogen with the 30:70 MeOH/acetonitrile system was observed. Injection of samples following an extraction procedure did not yield any peaks, indicating a lack of sensitivity for the UV detection system.